EP0756637A1 - Methods for calibrating chemical assays - Google Patents
Methods for calibrating chemical assaysInfo
- Publication number
- EP0756637A1 EP0756637A1 EP95914453A EP95914453A EP0756637A1 EP 0756637 A1 EP0756637 A1 EP 0756637A1 EP 95914453 A EP95914453 A EP 95914453A EP 95914453 A EP95914453 A EP 95914453A EP 0756637 A1 EP0756637 A1 EP 0756637A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- atp
- analyte
- caged
- assay
- derivative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Definitions
- the present invention relates to methods for calibrating chemical assays and more particularly but not exclusively to a method for calibrating assays for analytes of importance in analytical microbiology such as an assay for adenosine 5*- triphosphate (ATP) .
- ATP adenosine 5*- triphosphate
- Microbial biomass estimation is also important in a number of other applications, including control of waste processing, monitoring of sterilization processes and monitoring of air quality. In many cases, it is advantageous to be able to estimate the degree of microbial or chemical contamination within the shortest possible period of time.
- assay techniques are available for detection and quantification of analytes of interest, and examples include enzymic assays, enzyme linked immunosorbent assays (ELISA) , spectrophotometric assays, luminescence assays, fluorescence assays, chromatographic assays, assays based on measurement of changes in optical rotation of test sample, ion selective assays (including titrimetric assays) and colorimetric assays.
- the particular assay method used mainly depends upon the analyte to be detected and quantified.
- Standardization techniques are known which use radioactive isotopes for the purpose of standardizing the instrumentation rather than the assay per se .
- An example of such radioactive standardization is embodied in the Biolink Light Standard (Leaback, DH, Easy-to-use light standards as aids to luminometry, Szalay, A.A. et al (Eds) pp 33-37 - Bioluminescence and Chemiluminescence, Status Report. Proceedings of VII International Symposium of Bioluminescence and Chemiluminescence John Wiley & Sons, Chichester 1993) .
- Such standards are based upon gaseous - tritium - activated phosphors which emit light of a defined spectral range and exhibit predictable decay on long term storage. Such devices serve only to assist in calibration of the luminometers themselves.
- adenosine 5'-triphosphate is an important analyte in microbiological assays. ATP is found in live cells but not in dead cells and its presence in a sample can be indicative of microbial contamination.
- One widely used technique for assaying ATP is an ATP bioluminescence technique.
- ATP adenosine 5'-triphosphate
- AMP adenosine 5'-monophosphate
- PP- inorganic phosphate; C0 2 — carbon dioxide
- the amount of light produced by the reaction is directly proportional to the ATP concentration and can be detected using a sensitive light detector. This is the basis of the ATP-biolu inescence assay.
- Assays for ATP using the firefly luciferase reaction can be calibrated in two ways (Jago, P.H., Stanfield, G. Simpson, W.J. & Hammond, J.R.M. 1989. In ATP Luminescence: Rapid Methods in Microbiology, Society of Applied Bacteriology Technical Series, Vol. 26, Stanley P.E. et al. (eds) pp 53- 61) .
- the light output from reaction of the firefly luciferase reagent with the sample can be compared to that obtained from reaction of the firefly luciferase reagent with known amounts of ATP using a standard curve.
- the inherent activity of the firefly luciferase reagent itself may vary due to inconsistencies in production or handling. Even though the reaction' rate may be unaffected, there may be substances present in the reaction mixture which absorb the light produced to a significant extent, thus introducing error. Some substances, notably Zn 2+ , in addition to reducing the catalytic activity of firefly luciferase also change the wavelength of the light produced in the reaction. This can cause a reduced amount of light to be detected from the reaction in some types of lu inometers (Denburg, J.L. & McElroy, W.D. 1978. Arch . Biochem . Biophys . 141, 668-675) .
- step (ii) measuring a test property of the assay; (iii) exposing the sample/photosensitive derivative mix to a flash of visible light of pre-determined duration and intensity to release from the photosensitive derivative a known amount of analyte; (iv) re-measuring the test property; (v) repeating steps iii) and iv) inclusive from zero-n times as desired; (vi) calculating the change in the test property measurements; and (vii) using the calculated value(s) from step (vi) as a standard to determine the amount of analyte originally present in the sample.
- step (i) exposing a pre-determined amount of photosensitive derivative of an analyte to a flash of visible light of pre-determined duration and intensity to release from the photosensitive derivative a known amount of analyte; (ii) measuring a test property of the assay; (iii) repeating steps (i) and (ii) inclusive from zero-n times as desired; (iv) calculating the change in the test property measurements; and (v) using the calculated values from step (iv) as a standard against which results of an assayed sample can be compared.
- the photosensitive derivative is in solution prior to the photolysis step, although in some circumstances it may be desirable to use the photosensitive derivative in a dried state.
- the intensity and/or duration of the flash of visible light in step (iii) may be varied so as to compensate for a diminishing amount of photosensitive derivative available for photolysis.
- step (viii) calculating the change in the light emission measurements; and (ix) using the calculated value(s) from step (viii) as a standard to determine the amount of ATP originally present in the sample.
- the present method a non-radioactive, photostandardization technique, provides advantages over previously available standardization methods in respect of (i) precision and accuracy of results; (ii) verification of results; and (iii) user friendliness.
- a range of synthetic compounds of biological interest has been developed for use in physiological studies which contain a photo-sensitive chemical bond. When one of these compounds is exposed to a brief flash of intense light, a reaction product is released.
- the photo-sensitive compound is referred to as a *caged' compound, from which a molecule or ion of interest can be liberated.
- ATP is released from caged- ATP
- Ca 2+ is released from caged-Ca 2+ , and so on.
- Such photo- sensitive analyte derivatives are suitable for the present invention and their use, together with a high intensity photoflash lamp or Q-switched frequency-doubled laser provides a convenient and non-invasive means for standardizing a chemical assay.
- the finding that commercially available photoflash lamps can effect efficient release of analyte molecules from caged precursors is hitherto unknown.
- a predetermined amount of the 'caged' compound can be added to the sample to be assayed, or to the buffer used to dilute the sample to be assayed.
- a particular test property of the assay is then measured. The test property measured depends upon the assay in question but it could be emitted light, heat evolution, colour changes or other properties.
- a predetermined amount of the free compound is then released from the caged compound by exposing the mixture to a light flash of predetermined intensity and duration.
- the test property may then be re-measured and the change in test property measurements may be calculated.
- the calculated value may then be used as a standard to determine the amount of analyte originally present in the sample.
- a range of standard concentrations of the compound of interest can be produced by varying the degree of photolysis in a single strength solution of a 'caged 1 compound.
- a calibration series can be prepared, for example on a microtitre plate, without the need to pipette different volumes of standard analyte solutions.
- the conventional assay procedure could be followed but in selected assays, standardization could be achieved by photolysis of the 'caged' analyte prior to performing the assay step.
- this general photostandardization system can be used with a wide variety of assay systems, including enzyme linked immunosorbent assays (ELISA) , enzymic assays, spectrophotometric assays, fluorescence assays, assays based on measurement of changes in optical rotation of the test- sample, ion-selective assays (including titrimetric assays) and colorimetric assays.
- ELISA enzyme linked immunosorbent assays
- enzymic assays enzymic assays
- spectrophotometric assays spectrophotometric assays
- fluorescence assays assays based on measurement of changes in optical rotation of the test- sample
- ion-selective assays including titrimetric assays
- colorimetric assays colorimetric assays.
- a multi-point calibration rather than a single-point calibration.
- This can be achieved by generating a calibration series by separately photolysing different solutions of a single-strength 'caged' compound (ideally in a microtitre plate or its equivalent) .
- a degree of photolysis for example by employing one, two, three or four flashes of pre-determined intensity and duration, a set of calibration standards is obtained.
- the amount of analyte released with each sequential flash decreases.
- the flash intensity and/or duration it is possible to compensate for the diminishing amount of •caged' material available for photolysis, thereby resulting in production of equal quantities of analyte at each stage and generation of a linear calibration series.
- the amount of analyte released under a standard set of photolysis conditions can be determined by reference to a separately-prepared standard curve prepared using a non-derivatized form of the analyte.
- the concentration of photosensitive analyte in the reaction can be adjusted to achieve release of a certain amount of analyte, or the intensity and/or duration of the light flash can be adjusted to achieve the same end.
- Some materials phosphoresce when exposed to a high intensity flash of light, such as that produced by a flash gun.
- the intensity of such phosphorescence depends on the material.
- phosphorescence is a source of interference. Ways of minimizing or correcting for such interference include the following (note that more than one strategy can be used at any one time) .
- a first strategy is to introduce a time delay between the period of photolysis and the period of measurement.
- the logarithm of the intensity of the phosphorescence decreases in proportion to the increase in the logarithm of the time elapsed.
- a gap of a few seconds is sufficient to reduce the measured light emission to a value which is acceptable for many types of assays.
- a second strategy is to filter either the light used to irradiate the sample (or use monochromatic light such as that from a laser source) or to filter the light emitted by the cuvette material. Both strategies can be equally or differently efficacious, depending on the materials being used.
- a third strategy is to select materials which have a low potential to phosphoresce when exposed to the lighting conditions specified in the photostandardization protocol.
- a further strategy is to correct the result obtained after photolysis for the light emitted as a result of phosphorescence. This can be achieved using an electronic calculating device provided that (i) the inherent phosphorescence of the material is known, (ii) materials are selected to meet a standard specification and (iii) the time at which the measurements are made after exposure to light is standardized.
- 'caged' products are contaminated to a greater or lesser extent with 'uncaged' products.
- the presence of 'uncaged' products must be reduced to technically insignificant levels if optimal performance of the photostandardization technique is to be realized.
- contaminant ATP molecules can be eliminated (converted to ADP and AMP which do not react with firefly luciferase) by reacting the caged-ATP with an ATP-degrading enzyme such as apyrase either in a soluble or immobilized form.
- the luciferin-luciferase reagent containing the 'caged' ATP can be incubated for 8 - 20 hours or more at 4°C. During this time firefly luciferase catalyses hydrolysis of ATP but does not affect the level of caged ATP. ATP can also be removed using any of the techniques currently known in preparative chemistry including chromatography, selective precipitation, or extraction. Techniques of a similar nature can be used to treat 'caged' forms of other analytes prior to use for the purposes of photostandardizing assays using, in many cases, methods that are generally known. In some cases it may be desirable to further purify the 'caged' material.
- the commercially-available material consists of a pair of diastereoisomers, since the nitrophenyl group used as the photolabile 'cage' can be added to ATP in two possible configurations. While these stereoisomers have identical photochemical properties, in some applications, advantage may be obtained by the use of only one of the stereoisomers. For example, the interaction of each stereoisomer, or more specifically its ability to form inclusion complexes, with components of the assay reagent such as ⁇ -, ⁇ -, y-cyclodextrin will differ in the case of each stereoisomer.
- Some commerically-available firefly luciferase reagents contain cyclodextrins (see, for example, Lundin, A, Anson, J & Kau, P ATP extractants neutralised by cyclodextrins Campbell, A K et al (Eds) pp 399-402 in Bioluminescence and Chemiluminescence, Fundamentals and Applied Aspects, Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence, John Wiley & Sons, Chichester, 1994) . Stereoisomers of caged substances can be separated from one another using well-known techniques.
- a fixed amount of the 'caged* compound can be freeze-dried onto items of equipment used to peform an assay.
- items of equipment used to peform an assay examples include disposable pipette tips, disposable sample cuvettes, and disposable paper discs or strips.
- the 'caged* compound which has been freeze-dried onto the item, dissolves when a liquid such as a test sample or analysis reagent is brought into contact with it.
- a pre-determined amount of the analyte can be released from the 'caged' molecule at any time by photolysis, either when the 'caged* compound is in a dried state or in solution. This type of standardization strategy is of particular value in cases where the 'caged' compound lacks adequate stability in aqueous solution.
- analytes which can be standardized using one or more of the aforementioned strategies include ATP, adenosine onophosphate (AMP) , cyclic adenosine monophosphate (cAMP) , adenosine diphosphate (ADP) , guanosine triphosphate (GTP) , substrates for luminogenic reactions (such as D- luciferin) , inhibitors of luminogenic reactions (such as L- luciferin) , organic acids (such as oxalic acid) , fatty acids (such as arachadonic acid) , amino acids (such as phenylalanine) , sugars (such as glucose) , sugar phosphates (such as glucose-6-phosphate) , pesticides (such as atrazine) , naturally-occuring toxicants (such as ochratoxin A) , antibiotics (such as benzyl penicillin) , and compounds of pharmacological interest (such as dopamine, norepin
- ATP a photosensitive derivative of ATP
- ATP derivatives a nitrophenol ester of ATP which contains a photohydrolysable ester bond ( Figure 1) .
- Other members of this group of compounds could be substituted for the nitrophenol.
- a further example of a suitable ATP derivative is l-(4,5-dimethoxy-2-nitrophenyl) diazoethane (DMNPE) .
- Caged ATP has been used in a number of research applications relating to a variety of areas, including the study of muscle biochemistry. (Goldman, Y.E., Hibbard, M.G. McCray, J.A. & Trentha , D.R. 1982, Nature, 300, 701-705). It has not been previously suggested or used for the purpose of calibrating biochemical or microbiological assays.
- the yield of ATP from caged-ATP has been shown to be independent of pH over the pH range 6-9; furthermore, caged- ATP has been shown to be convenient to work with in that it is soluble in water at neutral pH and is not significantly photolyzed during several minutes exposure to subdued daylight (McCray, J.A. Herbette, L. Kihara, T. & Trentham, D.R. 1980, Proc. Natl. Acad. Sci., USA, 77, 7237-7241).
- a caged- ATP compound is incorporated into a firefly luciferase reagent at a pre-determined concentration.
- Firefly luciferase does not display any catalytic activity toward caged-ATP (see Example 1) , and therefore in the absence of added ATP, no light is produced from such a luciferase preparation.
- the reagent may be used to perform highly sensitive assays of ATP in the following way:-
- a sample and the reagent are mixed in a disposable luminometer cuvette.
- the sample may consist of an aqueous fluid of any origin or may be a specially prepared "extract" produced as a result of treatment with any of a variety of agents designed to release ATP from living organisms.
- the light emitted from the luminescent reaction is measured using a luminometer.
- a known amount of ATP is then released into the reaction mixture by exposing the cuvette together with its contents to a flash of visible light of high intensity, delivered from e.g. a photoflash lamp.
- the cuvette is then placed back into the luminometer and the light emission from the luciferase reaction measured once again. Since the light flash causes the release of a known amount of ATP from the caged-ATP present, the amount of light attributable to the presence of this known amount of ATP can be calculated by difference.
- the amount of ATP originally present in the reaction mixture can then be calculated.
- Caged-ATP (Calbiochem, USA, prod. no. 119127,5 mg) was dissolved in 0.5 ml sterile de-ionized water. The resulting stock solution was stored frozen at -20°C until required. Before use, it was diluted 1 in 1000 with sterile de-ionized water. To 3 ml of a commercially-available luciferase- luciferin reagent (Biotrace XT reagent, Biotrace Ltd., Bridgend, UK) was added 200 ⁇ l of sterile de-ionized water to form a control reagent or 200 ⁇ l of the caged-ATP stock solution to form a caged-ATP reagent. The reagents were then incubated for 1-6 hours at 20°C.
- sterile de-ionized water 300 ⁇ l was transferred to a clean disposable luminometer cuvette then 100 ⁇ l of control reagent or caged-ATP reagent added.
- the light emitted from the reaction was quantified using a Biotrace M3 Luminometer (Biotrace Ltd, Bridgend, UK) , which integrated the light response for 10 seconds after a 2 second delay.
- a volume of ATP solution (10 ⁇ l, 0.1 ⁇ M) was then added to the reaction mixture and the light output measured for a second time.
- caged-ATP might release ATP either by spontaneous hydrolysis, as a result of enzymic action, and/or by photolysis.
- the results shown in Example 1 indicate that release of ATP by the former two mechanisms is negligible and that, on exposure to a high intensity flash of light produced by a flashgun, ATP is produced rapidly from the caged-ATP present. Notes to Table l
- the high intensity photoflash (provided by a Hanimex 325A2 flash gun, Hanimex, UK) did not induce luminescence from empty cuvettes, nor from cuvettes containing test solutions such as water or caged-ATP, or luciferase-luciferin reagents in the absence of caged- ATP.
- the "ATP photostandard” (caged-ATP) produced 1.171 pmol ATP/assay on exposure to a high intensity photoflash.
- a luciferase/luciferin reagent which contained caged-ATP was prepared by adding caged-ATP to Biotrace MLX reagent (Biotrace, UK) to a concentration such that each 100 ⁇ l of reagent contained 43.1 pmol of caged-ATP (ie 431 nmol/1) . This reagent was incubated overnight at 4°C to reduce the background light emission.
- the assays were carried out as follows.
- ATP was added to a series of tubes (1 pmol per tube in 10 ⁇ l sterile de-ionized water) then 290 ⁇ l of trichloroacteic acid (TCA) solution in HEPES/EDTA buffer (original pH value of the buffer was 7.75) added.
- TCA trichloroacteic acid
- Luminescence was initiated in the case of each sample by adding 100 ⁇ l of the modified luciferase/luciferin reagent. The resulting light output was recorded in a Biotrace Multilite luminometer. The cuvette was then removed from the luminometer and exposed to a high intensity flash of light using a photoflash lamp which was mounted in a device of our own design (Serial number 005) . The cuvette was then placed back in the luminometer and the light produced by the reaction recorded for a second time.
- the ATP content of each tube was calculated on the assumption that each photolysis step resulted in formation of 1.15 pmol of ATP. All tests were carried out in duplicate.
- Caged-ATP (Calbiochem, USA) was dissolved in sterile de- ionized water to a final concentration of 14.4 ⁇ mol/1. Portions (300 ⁇ l) of the solution were transferred to clean disposable cuvettes then subjected to between one and 10 flashes using a Hanimex flashgun. After photolysis, the solutions were analysed by adding 100 ⁇ l of luciferase/luciferin reagent (Biotrace MLX reagent, Biotrace pic, UK) . The light emitted was quantified using a Biotrace Multilite luminometer which integrated the light signal for 10 seconds after a two-second delay.
- luciferase/luciferin reagent Biotrace MLX reagent, Biotrace pic, UK
- Phosphorescence of cuvettes can lead to significant light emission occurring from samples exposed to the lighting conditions used to photostandardize assays.
- the rate of decay of phosphorescence from such cuvettes is linear when plotted as a logarithmic plot.
- Caged-ATP solution (14.3 mmol/1; 10 ⁇ l) was added to 1 ml Lu it buffer (HEPES/EDTA/Mg buffer, pH 7.75; Lumac bv) containing 5 ⁇ l/ l of apyrase solution.
- the potato apyrase was a commercial preparation (So ase) obtained from Lumac bv and was reconstituted by adding 1 ml Lumit buffer to a vial of apyrase. At various times, 20 ⁇ l of this solution was transferred to clean disposable luminometer cuvettes containing 300 ⁇ l sterile de-ionized water. The light emitted by the sample was measured using a Biotrace Multilite luminometer (Biotrace, UK) .
- the cuvette was then removed from the luminometer and placed in a flash device of our own design. After receiving a single flash from a photoflash tube, the cuvette was returned to the luminometer and the light emission measured once again. Control experiments monitored the light output from reactions in which Lumit buffer was added to the sample of caged-ATP, rather than apyrase solution.
- the luminometer can not measure light outputs in excess of 1000000 RLU. Counts approaching this limit may behave in a non-linear way.
- a solution of caged-ATP (14.3 mmol/1; 10 ⁇ l) was transferred to 1 ml HEPES/EDTA/Mg buffer, pH 7.75 (Lumit buffer, Lumac bv) and 5 ⁇ l of a solution of potato apyrase added.
- the potato apyrase was a commercial preparation (Somase) obtained from Lumac bv and was reconstituted by adding 1 ml Lumit buffer to a vial of apyrase.
- the solution of caged-ATP containing the apyrase was incubated at 20°C for 40 min then diluted 1:1 with Lumit buffer.
- Portions (1 ⁇ l) of the solution were transferred to clean disposable plastic cuvettes (obtained from Biotrace pic) . This procedure resulted in each cuvette containing 143 pmol of caged-ATP.
- the cuvettes were then placed in a vacuum desiccator and freeze-dried at - 55°C under vacuum for two hours.
- the cuvettes were tested as follows. Sterile, de-ionized water (300 ⁇ l) was transferred to each cuvette then 100 ⁇ l luciferase/luciferin reagent (Biotrace MLX 24 reagent, Biotrace pic) added. The light emitted was recorded using a Biotrace Multilite luminometer, then ATP was released from the caged-ATP present using a photoflash lamp contained in a device constructed at BRF International (Box serial number 002) . The following results were obtained on replicate cuvettes,
- ATP could also be released from the freeze-dried material contained in the cuvettes by flashing, prior to addition of any samples or reagents to the cuvette. In performing such tests, the following results were obtained.
- Example 7 Preparation of a series of standard solutions of known concentration using caged-arachidonic acid' and varying quanta of light
- Caged-arachidonic acid (Molecular Probes Inc, USA; product number A-7062) is dissolved in HEPES/EDTA buffer (pH 7.75) to a final concentration of 14.4 ⁇ mol/1. Portions (100 ⁇ l) of the solution are transferred to clean disposable tubes then subjected to between one and 10 flashes using a Hanimex flashgun. After photolysis, the solutions are analysed using any method suitable for quantification of arachidonic acid, such as HPLC, GC/MS, TLC or specific enzymic methods.
- HEPES/EDTA buffer pH 7.75
- Portions (100 ⁇ l) of the solution are transferred to clean disposable tubes then subjected to between one and 10 flashes using a Hanimex flashgun. After photolysis, the solutions are analysed using any method suitable for quantification of arachidonic acid, such as HPLC, GC/MS, TLC or specific enzymic methods.
- a 'calibration curve' can be produced in this way which is analagous to that which could have been prepared by pipetting equal volumes of standard solutions, of different concentrations, into separate vials.
- the photostandardization technique as described allowed the same result to be achieved with substantial savings in operator effort.
- the technique is also non-invasive in nature.
- Caged-penicillin V (Molecular Probes Inc, USA: product number P-7061) is dissolved in HEPES/EDTA buffer (pH 7.75) to a final concentration of 14.4 ⁇ mol/1. Portions (100 ⁇ l) of the solution are transferred to clean disposable tubes then subjected to between one and 10 flashes using a Hanimex flashgun. After photolysis the solutions are analysed using any method suitable for quantification of penicillin V, for example using an ELISA test.
- a , 'calibration curve' can be produced in this way which is analagous to that which could have been prepared by pipetting equal volumes of standard solutions, of different 26 concentrations, into separate vials.
- the photostandardization technique as described allowed the same result to be achieved with substantial savings in operator effort. • ⁇ The technique is also non-invasive in nature.
- caged analytes which could be used in methods analagous to examples 7 and 8 include caged aspartic acid (Molecular Probes Inc USA: product number A-2505) , caged L-lysine (Molecular Probes Inc USA: product number B-7099) , caged L-epinephrine (Molecular Probes Inc USA: product number D-7057) , caged L-dopamine (Molecular Probes Inc USA: product number D-7064) , caged L-phenylalanine (Molecular Probes Inc USA: product number D-7093) and caged D-luciferin (Molecular Probes Inc USA: product number L-7085) .
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Application Number | Priority Date | Filing Date | Title |
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GB9406737A GB2288232A (en) | 1994-04-06 | 1994-04-06 | Photosensitive derivatives of ATP as assay control standards |
GB9406737 | 1994-04-06 | ||
PCT/GB1995/000794 WO1995027797A1 (en) | 1994-04-06 | 1995-04-06 | Methods for calibrating chemical assays |
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EP0756637A1 true EP0756637A1 (en) | 1997-02-05 |
EP0756637B1 EP0756637B1 (en) | 1998-12-02 |
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EP95914453A Expired - Lifetime EP0756637B1 (en) | 1994-04-06 | 1995-04-06 | Methods for calibrating chemical assays |
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US (1) | US5801007A (en) |
EP (1) | EP0756637B1 (en) |
JP (1) | JPH09511581A (en) |
CN (1) | CN1070920C (en) |
AT (1) | ATE174062T1 (en) |
DE (1) | DE69506394T2 (en) |
DK (1) | DK0756637T3 (en) |
ES (1) | ES2124542T3 (en) |
FI (1) | FI964003A (en) |
GB (3) | GB2288232A (en) |
RU (1) | RU2135586C1 (en) |
WO (1) | WO1995027797A1 (en) |
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US6117394A (en) * | 1996-04-10 | 2000-09-12 | Smith; James C. | Membrane filtered pipette tip |
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GB1604249A (en) * | 1977-05-31 | 1981-12-02 | Kolehmainen S | Selective measurement of somatic and microbial cells |
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EP0563858B1 (en) * | 1992-04-01 | 1997-07-09 | Nihon Millipore Kogyo Kabushiki Kaisha | Method of determining viable count |
US5366873A (en) * | 1992-06-03 | 1994-11-22 | Gideon Eden | Device and method for use in detecting microorganisms in a sample |
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1995
- 1995-04-06 WO PCT/GB1995/000794 patent/WO1995027797A1/en active IP Right Grant
- 1995-04-06 GB GBGB9620993.7D patent/GB9620993D0/en active Pending
- 1995-04-06 CN CN95193204.7A patent/CN1070920C/en not_active Expired - Fee Related
- 1995-04-06 RU RU96121570A patent/RU2135586C1/en active
- 1995-04-06 JP JP7526164A patent/JPH09511581A/en not_active Ceased
- 1995-04-06 US US08/721,984 patent/US5801007A/en not_active Expired - Fee Related
- 1995-04-06 AT AT95914453T patent/ATE174062T1/en not_active IP Right Cessation
- 1995-04-06 ES ES95914453T patent/ES2124542T3/en not_active Expired - Lifetime
- 1995-04-06 DE DE69506394T patent/DE69506394T2/en not_active Expired - Fee Related
- 1995-04-06 DK DK95914453T patent/DK0756637T3/en active
- 1995-04-06 EP EP95914453A patent/EP0756637B1/en not_active Expired - Lifetime
- 1995-04-06 GB GB9620993A patent/GB2301436B/en not_active Revoked
-
1996
- 1996-10-04 FI FI964003A patent/FI964003A/en unknown
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6210891B1 (en) * | 1996-09-27 | 2001-04-03 | Pyrosequencing Ab | Method of sequencing DNA |
US6258568B1 (en) | 1996-12-23 | 2001-07-10 | Pyrosequencing Ab | Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation |
Also Published As
Publication number | Publication date |
---|---|
US5801007A (en) | 1998-09-01 |
DK0756637T3 (en) | 1999-08-16 |
FI964003A0 (en) | 1996-10-04 |
CN1148870A (en) | 1997-04-30 |
RU2135586C1 (en) | 1999-08-27 |
GB2288232A (en) | 1995-10-11 |
ATE174062T1 (en) | 1998-12-15 |
GB2301436A (en) | 1996-12-04 |
ES2124542T3 (en) | 1999-02-01 |
JPH09511581A (en) | 1997-11-18 |
EP0756637B1 (en) | 1998-12-02 |
WO1995027797A1 (en) | 1995-10-19 |
DE69506394D1 (en) | 1999-01-14 |
GB9620993D0 (en) | 1996-11-27 |
DE69506394T2 (en) | 1999-08-26 |
GB2301436B (en) | 1998-02-18 |
GB9406737D0 (en) | 1994-05-25 |
FI964003A (en) | 1996-10-04 |
CN1070920C (en) | 2001-09-12 |
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